It is common to divide dynamic processes in live cells to one of three categories: diffusion of free or partly free proteins and inter-cellular entities; binding and unbinding of proteins to different inter-cellular entities; and structural changes to all or part of the cell. In general, each one of these categories describes a process that occurs in a different time scale, such as described in Wachsmuth et al., Genome organization: Balancing stability and plasticity. Biochimica et Biophysica Acta, 2008. 1783: p. 2061-2079.
Unbound molecules, such as a freely diffusing protein in a cell may travel a distance of micrometer in a fraction of a millisecond, whereas binding and unbinding of proteins may occur over a time range of between a millisecond to several seconds, and changes to cell structure typically happen over a time range of minutes to hours. Below we describe few of the most relevant methods for measuring the above-described processes in live cells. These methods, with very few exceptions, require labeling the studied proteins or structures with fluorescent dyes. Such methods are well developed through inserted fluorescent molecules, or through transfection of the cells with the DNA transcripts of fluorescent proteins that expresses the fluorescent proteins in the live cells.
Fluorescence correlation spectroscopy (FCS) is one common method for measuring motion of molecules while they bounce in and out of a small, defined region in a cell, as described in Ries, J. and P. Schwille, Fluorescence correlation spectroscopy. Bioassays, 2012. 34: p. 361-368. An entity, such as a molecule, may be labeled with a fluorescent dye and measured via fluorescence microscopy, where the size of an illuminated spot may be bound by the optics of the microscope used to measure it, and which may be limited by the point spread function of the microscope optics. The intensity of the illuminated spot may fluctuate and an autocorrelation function of the intensity may be calculated. The typical time that a particle remains within the small spot under measurement may correspond to the typical time that the autocorrelation function reduces to zero. This method may be applicable for measuring freely diffusing entities in the cell, where the diffusion coefficients are usually in the range of 1-100×10−4 μm2/sec. Although FCS may also be used for measuring binding processes, it may become complex and the extracted data may depend on the model that is being used for the analysis.
Another relevant method for measuring entity motion within a cell is fluorescence recovery after photobleaching (FRAP). In this method, both the diffusion and the binding properties of a selected protein may be measured. This method typically requires labeling the protein with a fluorescent dye. A sample of one or more cells may be initially measured. A high-intensity laser may then mark, or ‘burn’ the fluorescing molecules within a defined region in a technique also known as ‘bleaching’. The cellular sample may be repeatedly measured. If the sample in the bleached area recovers, one may extrapolate the typical time it takes the molecules to be replaced within the bleached area.
Continuous photobleaching (CP) is third method for measuring the motion of inter-cellular entities, and is typically simpler than FRAP.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the figures.